This invention provides novel benzodiazepinone compounds having drug and bio-affecting properties, their pharmaceutical compositions and method of use. In particular, the invention is concerned with arylacetamidoalanyl derivatives of 2,3-benzodiazepin-4-ones. These compounds uniquely inhibit xcex2-amyloid peptide (xcex2-AP) production, thereby acting to prevent the accumulation of amyloid protein deposits in the brain. More particularly, the present invention relates to the treatment of Alzheimer""s Disease (AD).
Alzheimer""s Disease is a progressive, neurodegenerative disorder characterized by memory impairment and cognitive dysfunction. AD is characterized pathologically by the accumulation of senile (neuritic) plaques, neurofibrillary tangles, amyloid deposition in neural tissues and vessels, synaptic loss, and neuronal death. It is the most common form of dementia and it now represents the third leading cause of death after cardiovascular disorders and cancer. The cost of Alzheimer""s Disease is enormous (in the U.S., greater than $100 billion annually) and includes the suffering of the patients, the suffering of families, and the lost productivity of patients and caregivers. As the longevity of society increases, the occurrence of AD will markedly increase. It is estimated that more than 10 million Americans will suffer from AD by the year 2020, if methods for prevention and treatment are not found. Currently, AD is estimated to afflict 10% of the population over age 65 and up to 50% of those over the age of 85. There is currently no effective treatment.
There have been many theories relating to the etiology and pathogenesis of AD. These theories were either based on analogies with other diseases and conditions (e.g., slow virus and aluminum theories), or based on pathologic observations (e.g., cholinergic, amyloid, or tangle theories). Genetic analysis can potentially differentiate between competing theories. The identification of mutations in the xcex2-amyloid precursor protein (xcex2-APP) of individuals prone to early onset forms of AD and related disorders strongly supports the amyloidogenic theories.
The xcex2-amyloid precursor protein (xcex2-APP), a large membrane spanning glycoprotein found in tissues of mammals, including humans, is encoded by a gene on the long arm of human chromosome 21. The main constituent of the plaques, tangles and amyloid deposits is known to be xcex2-amyloid peptides (xcex2-AP), composed of approximately 39 to 43 amino acid fragments of xcex2-APP, and in particular, the 40 amino acid fragment known as Axcex21-40. Several lines of evidence support the involvement of xcex2-AP in the pathogenesis of AD lesions. xcex2-AP and related fragments have been shown to be toxic for PC-12 cell lines and primary cultures of neurons, as well as causing neuronal degeneration with accompanying amnesia in rodents. Strong evidence for the role of xcex2-AP in AD consists of observations of genetic xcex2-APP mutations in individuals with certain forms of Familial Alzheimer""s Disease (FAD) and the correlation of disease onset with altered release of xcex2-AP fragments.
It is presently believed that the development of amyloid plaques in the brains of AD patients is a result of excess production and/or reduced clearance or removal of xcex2-AP. It is known that a basal level of xcex2-AP production may be a normal process and that multiple pathways for cleavage of xcex2-APP exist. Currently, however, it is unclear which classes of proteinases or inhibitors thereof that would be effective in treating AD. Various peptidergic compounds and their pharmaceutical compositions have been disclosed as useful in inhibiting or preventing amyloid protein deposits in brains of AD and Down""s Syndrome patients.
Dovey, et al. in European Patent Application 652,009, published May 10, 1995, disclosed a series of polyamido inhibitors of aspartic proteases, e.g. cathepsin D, for use in inhibiting intracellular xcex2-production.
A series of peptidergic compounds and their administration to patients to prevent abnormal deposition of xcex2-AP was disclosed by Cordell, et al., in WO 95/09838, published Apr. 13, 1995, as a means of treating AD.
Tamburini, et al. in WO 94/13319, published Jun. 23, 1994, disclosed methods for regulating formation of xcex2-AP by use of inhibitors of certain aspartic and serine proteases such as cathepsin D. Specifically, cathepsin D inhibitors were preferred. A series of peptidic compounds resembling pepstatin analogs was disclosed and claimed.
Xia, et al. disclosed substituted 2,3-benzodiazepin-4-ones (1) as being modulators of AMPA receptors for use in treating neurodegenerative disease (U.S. Pat. No. 5,891,871; WO9728135, WO9734878). 
R1 and R2 are, inter alia, hydrocarbon, carbocyclic, aryl and heteroaryl systems but are not alanylamido derivatives.
Audia, et al. have disclosed numerous peptidergic analogs of various azepinone heterocyclics, e.g. (2), but no 2,3-benzodiazepin-4-one derivatives were described. Audia""s compounds are reported 
to inhibit xcex2-amyloid formation (WO 9967221, WO 9967220, WO 9967219, WO 9966934, WO 9932453, WO 9838177, WO 9828268, WO 9822494, WO 9822493, WO 9822441, WO 9822433, and WO 9822430).
Nothing in these foregoing references can be construed to describe or suggest the novel alanyl derivatives of 2,3-benzodiazepin-4-ones of this invention or their use to inhibit xcex2-AP production.
A series of arylacetamidoalanyl derivatives of 2,3-benzodiazepin-4-ones have been synthesized. These compounds inhibit the production of xcex2-amyloid peptide (xcex2-AP) from xcex2-amyloid precursor protein (xcex2-APP). The pharmacologic action of these compounds makes them useful for treating conditions responsive to the inhibition of xcex2-AP in a patient; e.g., Alzheimer""s Disease (AD) and Down""s Syndrome. Therapy utilizing administration of these compounds to patients suffering from, or susceptible to, these conditions involves reducing xcex2-AP available for accumulation and deposition in brains of these patients.
The present invention comprises compounds of Formula I, their pharmaceutical formulations, and their use in inhibiting xcex2-AP production in patients suffering from or susceptible to AD or other disorders resulting from xcex2-AP accumulation in brain tissue. The compounds of Formula I include pharmaceutically acceptable acid and base salts, optical isomers (enantiomers) considered separately or as racemates, and solvates and/or hydrates thereof having the following formula and meanings.
The Formula I compounds are those having the following structure 
or a pharmaceutically acceptable salt or hydrate thereof wherein
R1 is hydrogen, hydroxyl, or oxygen;
R2 is C1-6 alkyl, C2-6 alkenyl or alkynyl, C3-7 cycloalkyl, C5-7 cycloalkenyl, aryl, heteroaryl, 
and 
with aryl being phenyl or naphthyl, and heteroaryl being furanyl, thienyl, or pyridinyl;
R3 and R4 are independently selected from C1-6 alkyl;
R5 and R6 are independently selected from hydrogen, C1-6 alkyl, C2-6 alkenyl or alkynyl, C3-7 cycloalkyl, C5-7 cycloalkenyl, 
and xe2x80x94CO2R10; or R5 can be taken with R4 or with R6 as a C3-6 alkanediyl chain thereby forming a fused ring system;
R7 is hydrogen or can be taken with R6 as xe2x95x90O;
R8 and R9 are independently selected from hydrogen, halogen, trifluoromethyl, C1-6 alkyl or alkoxy;
R10 is C1-6 alkyl;
n is zero or one;
m is one or two;
p is zero, one or two; and
the solid and dotted lines are either a single or a double covalent bond with the proviso that when they represent a double bond in the benzodiazepinone ring, n is zero.
There are three subclasses of compounds of Formula I. In subclass A, R6 and R7 are taken together as xe2x95x90O. In subclass B, R6 is selected from the listed moieties that define R6 while R7 is hydrogen. Subclass C has the ring double bond with n being zero.
While the term halogen can mean fluoro, chloro, bromo and iodo, fluoro and chloro are preferred. There are other structurally preferred groups. Phenyl, halo-substituted phenyl, and thienyl are preferred R2 groups.
It is understood that the stereochemistry of the asymmetric carbon being R3 is specified (see compound 1). Another asymmetric carbon is found in the benzodiazepinone ring where the nitrogen atom of the alanyl moiety is attached. There could also be other asymmetric centers, for example in certain of the substituent groups defining R2-R6.
As the compounds of the present invention possess asymmetric carbon atoms, the present invention includes the racemates and diastereomers as well as the individual enantiometric forms of the compounds of Formula I as described herein and in the claims. The use of a single designation such as (R) or (S) is intended to include mostly one stereoisomer. Mixtures of isomers can be separated into individual isomers according to methods which are known per se, e.g. fractional crystallization, adsorption chromatography or other suitable separation processes. Resulting racemates can be separated into antipodes in the usual manner after introduction of suitable salt-forming groupings, e.g. by forming a mixture of diastereosiomeric salts with optically active salt-forming agents, separating the mixture into diastereomeric salts and converting the separated salts into the free compounds. The possible enantiomeric forms may also be separated by fractionation through chiral high pressure liquid chromatography columns.
As indicated, the present invention also pertains to the pharmaceutically acceptable non-toxic salts of the basic centers of these compounds. Such salts include those derived from organic and inorganic acids such as, without limitation, hydrochloric, hydrobromic, phosphoric, sulfuric, methanesulfonic, acetic, tartaric, lactic, succinic, citric, maleic, sorbic, aconitic, salicyclic, phthalic, embonic, and enanthic acids.
The compounds of the present invention may be produced by the processes shown in Reaction Schemes 1, 2 and 3. In Schemes 1, 2 and 3 the symbols R1 to R7 and n are as defined supra. Scheme 1 illustrates a general process showing production of Formula I compounds of subclass A. Scheme 2 shows a general process for synthesis of Formula I compounds of subclass B while Scheme 3 gives a general process for synthesis of subclass C Formula I compounds.
The Scheme 1 process begins with the condensation of hydrazine VII with homophthalic anhydride to give the benzodiazepin-1,4-dione VIA which is converted to the amine IIIA by catalytic hydrogeneration of the azide precursor VA. Reaction of IIIA with an amino-protected alpha-amino acid IV (PG denoted a protecting group such as t-boc) provides the IIA intermediate which is coupled to the carboxylic acid intermediate X to yield the Formula IA product compound.
Scheme 2 illustrates a general synthesis of Formula IB products. Reaction of commercially available 3-isochromanones with suitably substituted hydrazines such as RNH-NHPG (PG denoted a protecting group such as t-boc) to give the alcohol XVII which is converted to cyclic compound VIB under intramolecular Mitsunobu reaction conditions. The amino functionality is introduced through azide VB and subsequent coupling of amine IIIB with an amino-protected amino acid IX provides the intermediate XII. The protecting group PG of XII is removed under acidic conditions to give Formula IIB product or treatment of IIB with an alkylating agent such as R-X to give Formula IB compound.
In Scheme 3 a general synthesis of Formula IC products is shown. Treatment of ortho-formylphenylacetic acid with R4-substituted hydrazine leads to formation of VIC which is converted to the amine intermediate IIIC by catalytic hydrogenation of the azide precursor VC. Reaction of VC with an amine-protected alpha-amino acid IV (PG is a protecting group) leads to the IIC intermediate which is coupled to the carboxylic acid intermediate X to yield the desired Formula IC product.
Greater experimental detail for preparation and reaction of the intermediates displayed in Schemes 1-3 is provided infra. One skilled in synthetic organic chemistry would be adept in modification of the presented synthetic methods in order to make compounds comprising Formula I.
Another aspect of the present invention involves the preparation of the novel compounds of Formula I. In that regard the novel process intermediates of Formula II are useful for the synthesis of products of Formula I.
The present invention also provides a method for the treatment or alleviation of disorders associated with xcex2-amyloid peptide, especially Alzheimer""s Disease, which comprises administering together with a conventional adjuvant, carrier or diluent a therapeutically effective amount of a compound of Formula I or a solvate or hydrate thereof.
In the method of the present invention, the term xe2x80x9ctherapeutically effective mountxe2x80x9d means the total amount of each active component of the method that is sufficient to show a meaningful patient benefit, i.e., improve one or more clinical parameters of disease activity, e.g. retention or cognition; or improve disease symptoms such as anxiety or neuromotor control. The subject amount is further characterized by inhibition of xcex2-amyloid peptide production as determined using in vitro assays or in vivo animal models of disease. When applied to an individual active ingredient, administered alone, the term refers to that ingredient alone. When applied to a combination, the term refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously. The terms xe2x80x9ctreat, treating, treatmentxe2x80x9d as used herein and in the claims means ameliorating one or more clinical indicia of disease activity in a patient having a disease associated with xcex2-amyloid peptide.
Accumulating findings have led to the perception that compounds that demonstrate inhibition of the formation of xcex2-AP from xcex2-APP would be clinically efficacious in treating disorders such as Alzheimer""s Disease, Down""s Syndrome, and certain forms of brain degeneration. In this regard, the compounds of the instant invention when administered orally or intravenously to experimental animals are capable of entering the CNS and inhibiting production of xcex2-AP formation in experimental animal models of disease.
Representative compounds of the instant series have been tested for their ability to inhibit xcex2-AP formation. Results of this screening assay are shown in Table 1.
Production and detection of Axcex2 made by cells in culture.
The use of transfected H4 (human neuroglioma) cells stably expressing constructs containing wild-type and variant forms of xcex2-APP have been used to identify inhibitors of Axcex2 production. Detection of Axcex2 can be shown by standard techniques such as immunoprecipitation of Axcex2 from the conditioned medium of 35S-methionine radiolabeled xcex2-APP-transfected cells such as previously described by Haass, et al., Nature, 359, p. 322-325 (1992) and Shoji, et al., Science, 258, p. 126-129 (1992) or detection of non-radioactively labeled Axcex2 by enzyme linked immunosorbent assay (ELISA ) as demonstrated by Seubert, et al., Nature, 359, p. 325-327 (1992). (The capture antibody is typically a mouse monoclonal (IgG1/kxcex2-APPa). The antibody recognizes the carboxyl terminal epitope of Axcex2. The specificity of the capture antibody insures measurement of Axcex2 without interference by other secreted xcex2-APP fragments that share amino acid sequence (Axcex21-16) with Axcex2 but lack the carboxy terminal region. The detecting antibody is typically an affinity purified rabbit polyclonal antibody, specific for the amino terminus of Axcex2. In the cell based assay conditioned medium from H4 cells is tested by ELISA for the amount of Axcex2 present. The cell based assay can been used to identify compounds that inhibit Axcex2 production. The assay is capable of detecting agents that inhibit cleavage at the xcex2-APP xcex2-secretase and/or the xcex3-secretase cleavage site as well as detecting any agent that interferes with the production and/or release of Axcex2.
A typical ELISA-based assay requires plating of the xcex2-APP transfected cells at a density sufficient for the rapid detection of the secreted Axcex2 (experimentally predetermined for a particular stable cell population) in a 96-well format. Cells are plated for at least 6 hours prior to the introduction of the test compound at which time the growth medium is replaced by fresh medium containing the agent to be tested. Test agents may typically consist of synthetic compounds, secondary metabolites from bacterial or fungal fermentation extracts, or culture supernatants from mammalian cells. All synthetic agents are initially screened at doses ranging from 10-100 xcexcM or in the case of extracts at sufficient dilution to minimize cytotoxicity. Incubation of the cells with the test agent will continue for approximately 16 hours at which time equal aliquots of media from each sample well is removed and placed into a previously prepared ELISA plate for Axcex2 quantitation. The ELISA is carried out in a manner described by others (Haass, et al., ibid; Harlow and Lane, Editors, Antibodies: A Laboratory Manual, Cold Spring Harbor Press, 1988) and the AD signal quantitated. Results are obtained by analysis of the ELISA plate following development and comparison to the mock treated cell populations and samples in which known amounts of Axcex2 were added to construct a standard concentration curve. A positive acting compound is one which inhibits cellular production of Axcex2 relative to levels in control samples by at least 50% at the initial tested concentration and does not show cytotoxicity. If a compound is found to be active then a dose response experiment is performed to determine the lowest dose of compound necessary to elicit the inhibition of the production of Axcex2.
Therefore, the compounds of Formula I or pharmaceutical compositions thereof are useful in the treatment of disorders associated with the aberrant extracellular deposition of amyloid, and (as formulated) will be effective to alter one or more clinical indicia of disease activity in a patient in need thereof.
In another embodiment, this invention includes pharmaceutical compositions comprising at least one compound of Formula I in combination with a pharmaceutical adjuvant, carrier or diluent.
In still another embodiment, this invention relates to a method of treatment of disorders characterized by aberrant extracellular deposition of amyloid and which are responsive to the inhibition of xcex2-amyloid peptide in a mammal in need thereof, which comprises administering to said mammal a therapeutically effective amount of a compound of Formula I or a nontoxic pharmaceutically acceptable salt, solvate or hydrate thereof.
In yet another embodiment, this invention relates to a method for treating systemic (vascular) amyloidosis, pulmonary or muscle amyloidosis, Alzheimer""s Disease, Down""s Syndrome, or other diseases characterized by extracellular amyloid deposition in a mammal in need thereof, which comprises administering to said mammal a therapeutically effective amount of a compound of Formula I or a non-toxic pharmaceutically acceptable salt, solvate or hydrate thereof.
For therapeutic use, the pharmacologically active compounds of Formula I will normally be administered as a pharmaceutical composition comprising as the (or an) essential active ingredient at least one such compound in association with a solid or liquid pharmaceutically acceptable carrier and, optionally, with pharmaceutically acceptable adjuvants and excipients employing standard and conventional techniques.
The pharmaceutical compositions include suitable dosage forms for oral, parenteral (including subcutaneous, intramuscular, intradermal and intravenous) bronchial or nasal administration. Thus, if a solid carrier is used, the preparation may be tableted, placed in a hard gelatin capsule in powder or pellet form, or in the form of a troche or lozenge. The solid carrier may contain conventional excipients such as binding agents, fillers, tableting lubricants, disintegrants, wetting agents and the like. The tablet may, if desired, be film coated by conventional techniques. If a liquid carrier is employed, the preparation may be in the form of a syrup, emulsion, soft gelatin capsule, sterile vehicle for injection, an aqueous or non-aqueous liquid suspension, or may be a dry product for reconstitution with water or other suitable vehicle before use. Liquid preparations may contain conventional additives such as suspending agents, emulsifying agents, wetting agents, non-aqueous vehicle (including edible oils), preservatives, as well as flavoring and/or coloring agents. For parenteral administration, a vehicle normally will comprise sterile water, at least in large part, although saline solutions, glucose solutions and like may be utilized. Injectable suspensions also may be used, in which case conventional suspending agents may be employed. Conventional preservatives, buffering agents and the like also may be added to the parenteral dosage forms. The pharmaceutical compositions are prepared by conventional techniques appropriate to the desired preparation containing appropriate amounts of the active ingredient, that is, the compound of Formula I according to the invention. See, for example, Remington""s Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 17th edition, 1985.
The dosage of the compounds of Formula I to achieve a therapeutic effect will depend not only on such factors as the age, weight and sex of the patient and mode of administration, but also on the degree of xcex2-AP inhibition desired and the potency of the particular compound being utilized for the particular disorder of disease concerned. It is also contemplated that the treatment and dosage of the particular compound may be administered in unit dosage form and that the unit dosage form would be adjusted accordingly by one skilled in the art to reflect the relative level of activity. The decision as to the particular dosage to be employed (and the number of times to be administered per day) is within the discretion of the physician, and may be varied by titration of the dosage to the particular circumstances of this invention to produce the desired therapeutic effect.
A suitable dose of a compound of Formula I or pharmaceutical composition thereof for a mammal, including man, suffering from, or likely to suffer from any condition related to aberrant production and/or extracellular deposition of xcex2-AP as described herein, generally the daily dose will be from about 0.05 mg/kg to about 10 mg/kg and preferably, about 0.1 to 2 mg/kg when administered parenterally. For oral administration, the dose may be in the range from about 1 to about 50 mg/kg and preferably from 0.1 to 2 mg/kg body weight. The active ingredient will preferably be administered in equal doses from one to four times a day. However, usually a small dosage is administered, and the dosage is gradually increased until the optimal dosage for the host under treatment is determined. In accordance with good clinical practice, it is preferred to administer the instant compounds at a concentration level that will produce an effective anti-amyloid effect without causing any harmful or untoward side effects. However, it will be understood that the amount of the compound actually administered will be determined by a physician, in the light of the relevant circumstances including the condition to be treated, the choice of compound of be administered, the chosen route of administration, the age, weight, and response of the individual patient, and the severity of the patient""s symptoms.
The following examples are given by way of illustration and are not to be construed as limiting the invention in any way inasmuch as many variations of the invention are possible within the meaning of the invention.
In the following examples, all temperatures are given in degrees Centigrade. Melting points were recorded on a Gallenkamp capillary melting point apparatus and are uncorrected. Proton magnetic resonance (1H NMR) spectra were recorded on a Bruker AC 300. All spectra were determined in the solvents indicated and chemical shifts are reported in xcex4 units downfield from the internal standard tetramethylsilane (TMS) and interproton coupling constants are reported in Hertz (Hz). Splitting patterns are designated as follows: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br, broad peak; dd, doublet of doublet; bd, broad doublet; dt, doublet of triplet; bs, broad singlet; dq, doublet of quartet. Infrared (IR) spectra using potassium bromide (KBr) or sodium chloride film were determined on a Perkin Elmer 781 spectrometer from 4000 cmxe2x88x921 to 400 cmxe2x88x921, calibrated to 1601 cmxe2x88x921 absorption of a polystyrene film and reported in reciprocal centimeters (cmxe2x88x921). Optical rotations [xcex1]D25 were determined on a Perkin-Elmer 41 polarimeter in the solvents indicated. Low resolution mass spectra (MS) and the apparent molecular (MH+) or (M-H)+ was determined on a Finnigan TSQ 7000. The element analysis are reported as percent by weight.
Synthesis of Intermediates
A. For IA Products